Minimizing thermally induced aggregation of DNase in solution with calcium

ABSTRACT

The present invention relates to the use of calcium ion and/or sugars to minimize thermal aggregation of DNase and to the use of calcium ion to stabilize liquid solutions of DNase, the solutions having a pH of less than neutral. DNase is the active pharmaceutical principle and the solutions may contain other pharmaceutically acceptable excipients making them suitable for pharmaceutical administration. In the first instance, calcium ion/sugar minimizes the effects of thermal aggregation in the solution. In the second aspect, calcium ion stabilizes the lower pH solutions from protein precipitation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.08/377,527, filed Jan. 20, 1995, now abandoned, which is a continuationof U.S. Ser. No. 08/206,504 filed Mar. 4, 1994, now abandoned. Thepresent application is related in subject matter to the disclosurecontained in U.S. patent application Ser. No. 07/448,038, filed Dec. 8,1989, now abandoned, and in U.S. patent application Ser. No. 07/289,958,filed Dec. 23, 1988, now abandoned, and in U.S. patent application Ser.No. 07/895,300, filed Jun. 8, 1992, now U.S. Pat. No. 5,279,823, and inU.S. patent application Ser. No. 08/206,020, filed on Mar. 4, 1994, nowabandoned. The content of these prior applications is hereby expresslyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention is related to results obtained from research onthe formulation of deoxyribonuclease, otherwise referred to as DNase, aphosphodiesterase that is capable of hydrolyzing polydeoxyribonucleicacid (DNA).

The present invention relates generally to the preparation of liquidsolutions of DNase that are protected from thermally induced aggregationof the DNase active principal component. The present invention relatesadditionally generally to the preparation of liquid solutions of DNasethat are maintained stable at pHs of less than neutral.

It relates to these solutions per se and to their methods of preparationand to their use clinically or for preparing further formulations usefulclinically in the treatment of disorders susceptible to the biologicalactivity of DNase, as discussed in more detail infra.

BACKGROUND OF THE INVENTION

DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleicacid. DNase has been purified from various species to various degrees.The complete amino acid sequence for a mammalian DNase was first madeavailable in 1973. See, e.g., Liao, et al., J. Biol. Chem. 248, 1489(1973).

DNase has a number of known utilities and has been used for therapeuticpurposes. Its principal therapeutic use has been to reduce theviscoelasticity of pulmonary secretions in such diseases as pneumoniaand cystic fibrosis, thereby aiding in the clearing of respiratoryairways. See, e.g., Lourenco, et al., Arch. Intern. Med. 142, 2299(1982); Shak, et al., Proc. Nat. Acad. Sci. 87, 9188 (1990); andHubbard, et al., New England Journal of Medicine 326, 812 (1992).

DNA encoding human DNase has been isolated and sequenced and that DNAhas been expressed in recombinant mammalian host cells, thereby enablingthe production of human DNase in mammalian commercially usefulquantities. See, e.g., Shak, et al., Proc. Nat. Acad. Sci. 87, 9188(1990). Recombinant human DNase (rhDNase) has been found to be usefulclinically, especially in purified form such that the DNase is free fromproteases and other proteins with which it is ordinarily associated innature.

The means and methods by which human DNase can be obtained inpharmaceutically effective form is described in the patent applicationscited above. Various specific methods for the purification of DNase areknown in the art. See, e.g., Khouw, et al., U.S. Pat. No. 4,065,355,issued Dec. 27, 1977; Markey, FEBS Letters 167, 155 (1984); and Nefsky,et al., Euro. Journ. Biochem. 179, 215 (1989).

The present application is predicated on the use of such DNase forformulation. DNase can be employed as such, as a mixture of deamidatedand non-deamidated forms, or in isolated deamidated and non-deamidatedforms. The preparation and separation of such forms are the subjectmatter of a patent application cited above.

The present invention is directed to the preparation of liquid solutionsof DNase (including all of its biologically active forms as previouslynoted) that are stable to thermally induced aggregation of DNase.

The present invention is directed to the preparation of stabilizedliquid solutions of DNase (including all of its biologically activeforms as previously noted). These liquid solutions containing DNase insubstantially non-deamidated form are maintained at pHs of less thanneutral in stable form such that precipitation of material does notoccur to any substantial extent, and therefore, the solutions are in aclear form suitable for pharmaceutical administration. Such less thanneutral pH levels result in a reduction of the rate of deamidation ofthe DNase principle during storage. At storage at elevated temperatures(upwards of 37° C.), such lower pH solutions result in precipitationproducts. The present invention, in an aspect, stabilizes such solutionsfrom such precipitation.

SUMMARY OF THE INVENTION

The present invention is predicated upon the finding of an exceptionalcharacteristic found attributable to a particular component which inliquid solution together with DNase as biologically active principle,protects said DNase from thermally induced aggregation. This particularcomponent in liquid solution together with DNase as biologically activeprinciple protects and stabilizes the solution from precipitationeffects resulting in clear solutions which are suitable forpharmaceutical administration.

The pharmaceutical specifications permitting the storage with suitableshelf life of DNase requires the retention of at least 80% biologicalpotency at from 2 to 8° C. over a sustained period of time. Therecommended pH of such solutions are about neutral, more particularlyapproximately 6.5. It has been discovered that at this pH range,deamidation occurs at a relatively constant rate resulting in solutionsin which the DNase component is increasingly deamidated. The deamidationtakes place at the asparagine residue that occurs at position 74 in theamino acid sequence of native mature DNase. Attention is directed toU.S. Ser. No. 07/895,300, filed Jun. 8, 1992. In that applicationattention is focused on the separation of deamidated and non-deamidatedDNase from one another for separate formulation into pharmaceuticallyadministrable forms.

In this aspect of the present invention, it has been found that loweringthe pH of such liquid solutions containing DNase substantially reducesthe rate constant of deamidation resulting in liquid formulations whichare relatively stable as to deamidation and thus the DNase remains inits non-deamidated form which is biologically more potent. However, thereduction of pH results in a by-process of precipitation of materialsfrom the liquid solutions when stored at about 37° C., which isunacceptable from a pharmaceutical formulation standpoint.

The present invention in this aspect relates to the stabilization ofsuch less than neutral pH liquid solutions containing DNase fromprecipitation resulting in solutions that are pharmaceuticallyadministrable.

The introduction and use of calcium ion (Ca⁺2) protects such less thanneutral pH liquid solutions of DNase from precipitation resulting inclear solutions that are suitable for pharmaceutical storage andadministration, without the need for refrigeration. Thus, the presentinvention in this aspect relates to a method of stabilizing liquidsolutions containing DNase as active principle, such solutions being ata pH of less than neutral such that deamidation is deterred or inhibitedwhich comprises employing amounts of calcium ion in said solution thatprotect such solutions from precipitation resulting in liquidformulations of DNase that are suitable for storage and ultimatepharmaceutical administration. These solutions are maintained in astable form without any or any substantial precipitation that otherwiseoccurs at pHs of less than neutral. Further, these solutions result in aminimal deamidation of the active component of DNase.

In the alternative aspect of the present invention, in signaldistinction from certain other divalent cations, calcium ion (Ca⁺²)additionally protects DNase from thermally induced aggregation in liquidsolution. Thus, the present invention relates to a process of minimizingthermally induced aggregation of DNase in liquid solution comprisingDNase as active principle which comprises employingDNase-aggregation-minimizing amounts of calcium ion in said solution.

In a further embodiment of this alternative aspect of this invention,protection of DNase from thermally induced aggregation in liquidsolution is obtained by the addition of sugars to said solutions, eitherin lieu of or additional to the presence of calcium ion.

Thus, the present invention is directed to methods for the preparationof liquid solutions comprising DNase as active principle whichcomprising using in said solutions an amount of calcium ion and/or sugarthat: 1) minimizes DNase aggregation brought about from thermalinstability (a DNase-aggregation-minimizing amount) and an amount ofcalcium ion that 2) stabilizes said solutions that are at a pH of lessthan neutral from precipitation resulting in said solutions being clearand thus in a form suitable for storage and pharmaceuticaladministration. In the latter, deamidation of the DNase is inhibited, aconsequence of the lowered pH (from neutral).

Stated another way, the present invention is directed to a method forthe preparation of a liquid solution comprising DNase as activeprinciple which comprises utilizing in said solution an amount ofcalcium ion and/or sugar that stabilizes said solutions from boththermally induced DNase aggregation and an amount of calcium ion thatstabilizes said solutions from precipitation effects when said solutionsare at less than neutral pH. In the latter instance, deamidation of theDNase is inhibited, a result of the lowered pH of the solution.

The present invention is further directed in different aspects to thesolutions themselves comprising DNase as active principle and amounts ofcalcium ion, and to the use of these solutions for the treatment ofdisorders in which the biological activity of DNase can be exploited. Aswell, it is directed to methods for the use of such solutions in thepreparation of further formulations comprising DNase as active principlesuch as subjecting said solutions to elevated temperatures, e.g. as inspray-drying techniques to produce pharmaceutically acceptableformulations of DNase in the form of a respirable DNase-containingpowder, suspension or solution that is therapeutically effective whenadministered into the lung of an individual. Further, such solutionsthat are at less than neutral pH, in order to inhibit deamidation of theDNase, are rendered stable to (lower pH) induced precipitation whenstored at temperatures at about ambient temperature or above.

The present invention is further directed to all associated embodimentsthereof relating to the preparation and use of liquid solutions ofDNase, which is essentially in monomeric form and/or inhibited indeamidation, and to stabilize against precipitation at pHs of less thanneutral, employing the calcium cation to minimize thermal aggregation ofthe DNase.

The liquid solutions hereof comprise as essential components DNase asthe biologically active principle and a source of calcium cation. Insuch solutions the DNase is in substantially monomeric form, and wheresuch solutions are at a pH of less than neutral, deamidation of theDNase is inhibited, and in those instances, the solutions are stabilizedagainst precipitation when stored at ambient or elevated temperatures.

The calcium ion source can be virtually any calcium salt supplieddirectly or formed in situ from a suitable calcium source that ispharmaceutically acceptable such as calcium chloride, in its varioushydrated and anhydrous forms, calcium oxide and calcium carbonate. Thecalcium source for the calcium cation active component of the presentliquid solutions of DNase is present generally at a concentration offrom about 1 mM to about 1 M, and more preferably, from about 10 mM toabout 100 mM.

The sugars may include, for example, α-lactose monohydrate, mannitol,trehalose, sucrose, and the like. They are employed in concentrationsgenerally of from about 50 to about 200 mg/ml., although concentrationsoutside of this range can also be used.

The solutions hereof may contain other components, such as excipients,with the only requirements being that such other components arepharmaceutically acceptable and do not interfere with the effect ofcalcium cation.

The solutions hereof can be used as such, or they can be used for thepreparation of pharmaceutically acceptable formulations comprising DNasethat are prepared for example by the method of spray-drying the liquidsolutions hereof and collecting the spray-dried product as a dispersibleDNase-containing powder that is therapeutically effective whenadministered into the lung of an individual. The calcium ion protectingeffect applies to any situation in which rhDNase solutions are exposedto elevated temperature and/or where such solutions are at a pH of lessthan neutral.

For spray-drying the latter embodiment, attention is again directed tothe co-pending U.S. application Ser. No. 08/206,020 filed Mar. 4, 1994for the details concerning the spray drying procedure.

Present results would indicate that minimization of thermal-inducedaggregation of DNase in solution can be achieved using lowertemperatures in the range less than about 60° C. and proteinconcentrations of about less than 1 milligram per milliliter, with a pHof about 6 to 7 and a concentration of excipient of more than about 10millimoles (at the concentration of DNase given above).

Present results would indicate that minimization of precipitation ofDNase solutions at a pH of less than neutral (in which solutionsdeamidation of the DNase is inhibited) results in solutions that can bepharmaceutically administered where such solutions are stable over longperiods of time, remaining clear for ultimate such pharmaceuticaladministration.

The DNase formulations hereof are employed for enzymatic alteration ofthe viscoelasticity of mucus within the lung. Such formulations areparticularly useful for the treatment of patients with pulmonary diseasewho have abnormal viscous, purulent secretions and conditions such asacute or chronic bronchial pulmonary disease, including infectiouspneumonia, bronchitis or tracheobronchitis, bronchiectasis, cysticfibrosis, asthma, tuberculosis and fungal infections and the like. Forsuch therapies, the novel formulations hereof are instilled generally bymethods familiar to those skilled in the art into the bronchi of theindividual being treated. The formulations hereof are particularlysuited for the assured introduction into the lung of DNase such that atherapeutically effective amount of DNase is delivered to the individualby direct action in the lung.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a size exclusion chromatogram of rhDNase. The sample wasprepared from a solution of 4.7 mg/ml DNase, 150 mM sodium chloride, 1mM calcium chloride, pH 6.7, heated at 65° C. for 60 seconds.

FIG. 2 shows the protective effect of calcium ion against thermallyinduced aggregation of rhDNase in solution at 65° C. (4.7 mg/ml rhDNase,150 mM sodium chloride, pH 6.5 to 6.7).

FIG. 3 shows an SDS-PAGE gel of thermally induced aggregated rhDNase(4.7 mg/ml rhDNase, 150 mM sodium chloride, 1 mM calcium chloride, pH6.7). The normal molecular weights of the marker proteins are indicated.

FIGS. 4a-d are differential scanning calorimetry (DSC) thermograms ofrhDNase showing the effects of sugars at various concentrations.

FIGS. 4e-h shows the denaturation or melting temperature (Tm) andenthalpy (Hm) of rhDNase plotted versus sugar concentration.

FIG. 5 shows the effect of pH on rates of deamidation of rhDNase at 37°C. by tentacle ion-exchange chromatography.

FIG. 6 shows the effect of calcium on stability of rhDNase: 1 mg/ml, 2ml in 5 cc glass vial with siliconized Teflon stopper at 25° C. storage.

FIG. 7 illustrates the kinetics for deamidation of rhDNase: 50 mg/ml in150 mM NaCl, 1 mM CaCl₂, pH 5 and 6.

FIG. 8 shows pH variance of rhDNase: 50 mg/ml in 150 mM NaCl, 0-200 mMCaCl₂, pH 5 at 37° C.

FIG. 9 demonstrates the physical stability of rhDNase, 50 mg/ml in 150mM NaCl, 0-200 mm CaCl₂, pH 5 at 37° C.

FIG. 10 shows the physical stability of rhDNase, 50 mg/ml in 1 mM NaOAcbuffers, pH 5 and 5.2 at 37° C.

FIG. 11 shows pH of 50 mg/ml rhDNase in 1 mM acetate buffers 1-100 mMCaCl₂, isotonic with NaCl at pH 5 and 5.2, 37° C.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

By the term “DNase” or “human DNase” or “recombinant human DNase” orgrammatical equivalents herein is meant a polypeptide having the aminoacid sequence of human mature DNase as well as amino acid sequencevariants thereof (including allelic variants) that are enzymaticallyactive in hydrolyzing DNA. Thus, the terms herein denote a broaddefinition of those materials disclosed and prepared in the variouspatent applications cited above and incorporated herein by reference. Itwill be understood that the terms include both purified mixtures ofdeamidated and non-deamidated human DNase as well as purified forms ofeach.

By the term “excipient” herein is meant a pharmaceutically acceptablematerial that is employed together with DNase for the proper andsuccessful preparation of a spray-dried formulation that results intherapeutic effect when administered into the lung of an individualpatient. Suitable excipients are well-known in the art, and aregenerally described above and, for example, in the Physician's DeskReference, the Merck Index and Remington's Pharmaceutical Sciences.

By the term “therapeutically effective” and grammatical equivalentsthereof herein is meant dosages of from about 1 microgram to about 1milligram of human DNase per kilogram of body weight of the individualbeing treated, administered within the pharmaceutical formulationshereof. The therapeutically effective amount of human DNase will depend,for example, upon the therapeutic objectives and the condition of theindividual being treated. In all of that, the present invention providesas an essential component, formulations containing therapeuticallyeffective amounts, the formulations being prepared such that theysuitably provide such therapeutic effect when administered into the lungof the individual.

B. Preferred Embodiments/Examples

1. Thermally Induced Aggregation

a. Calcium Ion

Materials

Stock solution: Recombinant Human DNase (rhDNase), 4.7 mg/ml, originallyformulated in 150 mM NaCl and 1 mM CaCl₂, pH 7.0±1.0 was used as it isor adjusted as described.

Methods

Thermally induced aggregation was carried out by heating rhDNasesolutions in 3 cc glass vials for lyophilization which werepre-equilibrated at the set temperatures in a water bath (FisherScientific). The temperature was controlled by a water circulator(Isotemp Immersion Circulator Model 730 Fisher Scientific) within anaccuracy of ±0.2° C. as measured by a temperature probe (ThermistorThermometer, Omega Engineering, Inc.). Solutions of rhDNase werepipetted into the vials, heated for different lengths of time, andtransferred to an ice-bath to terminate any thermally induced reactions.

Size exclusion chromatography (SEC) was employed to quantify the amountof monomer and aggregates. When precipitation occurred (e.g., in casesof prolonged heating at high temperatures), the solutions were filtered(0.22 μm pore-size filters, ultra-low protein binding, Millex-GV). Forthe filtered samples, the % monomer or aggregate was corrected for theprecipitated protein using the peak area of the control (i.e. time zero)sample. Depending on the concentrations, the solutions were then dilutedwith the SEC mobile phase to 1 mg/ml rhDNase for the measurement. Therunning conditions were:

Mobile phase: 5 mM HEPES, 150 mM NaCl, 1 mM CaCl₂.2H₂O, adjusted to pH7.0 with NaOH. The absorbance was measured at 280 nm. Injection volumeis 100 μl (1 mg/ml). Flow rate 1.0 ml/min. Running time was 20 min.

The amount of aggregates was expressed as a fraction of the peak areaeluted at 5.3 min to the total area at 5.3 min and 7.1 min (FIG. 1). Thepeak areas were automatically integrated by the computer. In case ofunsatisfactory integration (e.g. baseline shift), manual integration wasemployed.

EXAMPLE 1

Initial Study to Establish that Aggregation Takes Place Predominantly inSolution Rather than in the Solid State

Stock solutions of rhDNase, after dialysis in water to remove excesssalts, were either i) heated at 70-80° C. for 5 min or ii) lyophilized.The lyophilized powders were then either i) heated in the vial at 70-80°C. for 30 min or ii) pressed on a hot-plate at 80° C. for differentlengths of time up to 3 min. The samples were reconstituted in water forthe SEC measurements.

EXAMPLE 2

Effect of Calcium and Other Divalent Cations

Calcium and other divalent cations are known to stabilize bovine DNase Iagainst denaturation. [Paulos et al., The Journal of BiologicalChemistry 247, 2900 (1972)] The possible stabilizing effect of the ionon the thermal aggregation of rhDNase was studied at 65° C.Predetermined amounts of calcium chloride were dissolved in the stockrhDNase solutions (pH 6.7) to give higher calcium concentrations of 9and 106 mM as compared to 1 mM in the original solution.

For the other divalent cations, known amounts of analytical grade ZnCl₂,MnCl₂.4H₂O, and MgCl₂.6H₂O were respectively dissolved in the DNasestock solution to give a concentration of 100 mM of the cations.

The study was carried out at 65.3±0.20° C. As precipitation occurred insome of the solutions, those samples were filtered (0.22 μm filter unit,Millex-GV 4) before SEC analysis.

Chemical Nature of the Soluble Aggregates

The chemical nature (covalently bond or disulfide linkage) of theaggregates was analyzed by SDS-PAGE (Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis). Two fully aggregated samples weremeasured. A 10 μg (in 10 μl) of 1 mg/ml rhDNase load was used. The gelswere stained with Coomassie blue.

Thermally Induced Aggregation of rhDNase in the Solution and Solid-state

Table 1 shows that heating the rhDNase powders results in less than 1%aggregated rhDNase whereas in solution the protein can fully aggregatequite rapidly. Thus, thermally induced aggregation occurs much moresignificantly in solution than in the solid state. This suggests thatthe aggregates found in the spray-dried DNase powders were formed in thesolution state in droplets or while the powder still contains a highmoisture content.

Effect of Calcium and other Cations (at pH 6.4±0.2)

Divalent cations were reported to affect the stability of bovine DNasestructure.

FIG. 2 and Table 2 indicate the effect of calcium ions. At 9 mM CaCl₂,the protection against thermal aggregation at 65° C. for 5 min is 98%;at 100 mM CaCl₂ it offers complete protection. Other divalent cationssuch as Mn²⁺, Mg²⁺ and Zn²⁺ (chloride as the same anions) at 100 mM didnot give such protection as Ca²⁺. In fact, Zn²⁺ precipitates the proteineven at room temperature (Table 3). Thus, rather than an ionic strengtheffect, calcium ions appear to specifically bind to the proteinpreventing aggregation.

Chemical Nature of the Aggregates

The SDS-PAGE results (FIG. 3) show that the band pattern of theaggregated samples are the same as the non-aggregated ones, indicatingthat the aggregates dissociate in the SDS. Thus, the aggregates are notcovalent or linked by disulfide bond.

TABLE 1 Thermally Induced Aggregation of rhDNase in the Solution andSolid-state Sample % Monomer Solution, Control¹ 99.92 Solution,Lyophilized² 99.62 Solution, Heated & Lyophilized³ 0.88 Solution,Lyophilized & Heated⁴ 98.96 Solution, Lyophilized & Heated⁵ 99.33Solution, Lyophilized & Heated⁶ 99.02 Solution, Lyophilized & Heated⁷99.01 Notes: ¹Solution after dialysis in water as a control with nofurther treatment ²Solution 1 lyophilized to powder ³Solution 1 heatedto 70-80° C. for 5 min, then lyophilized ⁴Lyophilized powder of 2 heatedto 70-80° C. in a water bath for 30 min ⁵Lyophilized powder of 2,pressed on a hot-plate at 80° C. for 30 sec ⁶Lyophilized powder of 2,pressed on a hot-plate at 80° C. for 1 min ⁷Lyophilized powder of 2,pressed on a hot-plate at 80° C. for 3 min.

TABLE 2 Effect of Calcium Ion on the Thermally Induced Aggregation ofrhDNase in Solutions at 65° C. (4.7 mg/ml, 150 mM NaCl, pH 6.6 ± 0.1 %Monomer Time (s) 1 mM Ca²⁺ 9 mM Ca²⁺ 106 mM Ca²⁺ 0 (control) 99.64 100.099.97  30 98.96 99.92 99.98  60 91.14 99.95* 99.96  90 79.16 99.78 99.90120 69.05 99.45 99.81 180 51.87 99.25 99.96 300 36.63 98.17 99.95

TABLE 3 Effect of Divalent Cations on the Thermally Induced Aggregationof rhDNase in Solutions (4.7 mg/ml rhDNase, 150 mM NaCl, 1 mM CaCl₂, pH6.2-6.5) at 65° C. % Monomer Time (s) Mn²⁺ Mg²⁺ Zn²⁺ 0 99.99 99.05 <19(ppt'd) (control) 30 99.57 — — 90 <70 (ppt'd) — — 120 <47 (ppt'd) <74(ppt'd) — 180 <31 (ppt'd) <73 (ppt'd) — Note: the % monomer in theprecipitated (ppt'd) samples were only estimated values as the monomerpeak of the control samples went offscale in the chromatogram. Theactual % monomer would therefore be even lower. These results should becompared to the low and high calcium effects in the previous table(Table 2).

b. Sugars

Materials and Methods

A stock rhDNase solution of 20 mg/ml was prepared from a startingsolution (rhDNase 6.4 mg/ml, 150 mM NaCl, 1 mM CaCl₂) by concentrationin an Amicon deafiltration cell followed by dialysis in Milli-Q water toremove the salts. The solutes (additives) which were studied for theireffects on thermal stability of rhDNase were: α-lactose monohydrate(Sigma, lot 72H0563), mannitol (Sigma, lot 31H0181), trehalose (Sigma,lot 112H3903), sucrose (AR, Mallinckrodt, lot 8360 KBTA).

Differential Scanning Calorimetry (DSC)

DSC was carried out on a highly sensitive differential scanningcalorimeter (DSC 120, Seiko Instruments) which has a detectability <2μW. The solutions were sealed in a silver sample pan (60 μl) with waterused as the reference. Preliminary runs were done on a series of purerhDNase solutions using scanning rates ranging 0.1-2.0° C./min.Generally, a fast heating rate will result in a broader peak with ashift in the baseline, while slow heating will give a low signal tonoise ratio. With regard to the protein concentration, while lowconcentration will not give sufficient DSC signal, high concentrationwill facilitate aggregation. The optimal protein concentration andheating rate were found to be around 10 mg/ml and 1.2° C./min,respectively, which were subsequently employed throughout this study.

Preparation of rhDNase Solutions Containing Additives

Aqueous solutions of different additives were prepared by dissolving aknown amount of the solute in Milli-Q water. The solution pH wasadjusted using diluted NaOH or HCl to a value between 6 and 7 (this willgive rise to a difference within 1.0° C. in the T_(m) and 0.5 J/g inH_(m), see Results and Discussion). 70-100 μl of the 20 mg/ml rhDNasestock solution were then mixed with an equal volume of additivesolutions (which was substituted by pure water for the control rhDNasesamples). The final pH of the solutions was measured as 6.3 to 7.0. Nobuffer was added in order to avoid possible interactions of the bufferspecies with rhDNase and/or additives.

Proteins unfold or ‘melt’ at elevated temperatures and usually involveendothermic heat changes. For rhDNase, the apparent denaturationtemperature (T_(m)) and enthalpy (Hm) of 10 mg/ml rhDNase in pure waterat pH 6.8 were found to be 67.4±0.3 and 18.0±0.2 (n=4), respectively,values typical of globular proteins.

Effects of Sugars

FIGS. 4a-d show the protective effects of different sugars(disaccharides: lactose, sucrose and trehalose,monosaccharide; mannitol)on rhDNase against thermal denaturation. In general, both the T_(m) andH_(m) appear to increase monotonically with the sugar concentration(FIGS. 4e-h). The effects could be explained thermodynamically. Additionof sugars (as stabilizers) increases the chemical potential of theprotein (both native and denatured state). However, because of thelarger surface area of contact between the unfolded protein and solvent,the increase is higher (and thus more favorable) for the denatured statethan the native state. Thus, the native state is stabilized becausethermodynamically it will be more unfavorable to move to the denaturedstate.

2. Liquid Solution Stabilization

The major route of degradation for rhDNase is deamidation. Deamidationcorrelates with loss in activity as assessed by the methyl greenactivity assay. The rate of deamidation was also found to be highlydependent on the pH of the formulation (FIG. 5). In particular, as thepH decreases, the rate of deamidation can be minimized even at higherstorage temperatures.

The importance of Ca⁺⁺ in the stabilization of rhDNase was demonstratedin an experiment in which rhDNase 1 mg/mL in 150 mM NaCl, 1 mM CaCl₂)was treated with 1 mM EDTA to remove the exogenous Ca⁺⁺ bound to theprotein, and formulated into phosphate buffer at pH 6. Analysis of Ca⁺⁺indicated there were still 1 to 1.5 moles Ca⁺⁺/mole of rhDNase. Theactivity of rhDNase in this formulation was shown to be less stable thanthe original formulation in 1 mM CaCl₂as shown in FIG. 6. The percentdeamidation was about the same for both formulations suggesting that thedecrease of activity was not related to different rates of deamidationin the formulation (see inset, FIG. 6). The conclusion was that PO₄ wascompeting with rhDNase for calcium resulting in a less stable protein.As the bulk rhDNase in current formulation is being concentrated, theratio of Ca⁺⁺ to rhDNase decreases accordingly. Therefore, it wasreasoned that supplementing the current formulation with increasingamounts of Ca⁺⁺ may enhance the stability of rhDNase at higherconcentrations.

Materials & Methods

Preparation of 50 mg/mL rhDNase in 150 mM NaCl. 1 mM CaCl₂ pH 5 & 6. byAcid Titration

rhDNase, (4.7 mg/mL, 150 mM NaCl, 1 mM CaCl₂) was concentrated to ˜50mg/ML at 2-8° C. by Amicon ultrafiltration using a YM10 membrane. Theconcentrated rhDNase solution was divided into two equal aliquots. Thefirst aliquot was adjusted to pH 6 with 1N HCl. The second aliquot wasadjusted to pH 5. Both solutions were filtered through a 0.2 μm filterunit and sterile filled into 3 cc glass vials with a nominal volume of 1mL. The samples were stored at −70°, 2-8°, 15°, 25° and 37° C. Thestability of these rhDNase solutions was assessed over 6 months bycolor/clarity, pH, UV for protein concentration, methyl green (MG)activity assay, size-exclusion chromatography for aggregates, andtentacle ion-exchange chromatography for percent deamidation. SDS-PAGEwas performed initially to confirm the presence of aggregates.

Preparation of 50 mg/mL rhDNase in 150 mM NaCl. 0-200 mM CaCl₂. pH 5. byAcid Titration

The high concentration rhDNase formulation was prepared as describedabove. After concentration the rhDNase was dialyzed into solutionscontaining 150 mM NaCl, 10-200 mM CaCl₂, at 2-8° C. for 36 hours withtwo buffer changes. Two aliquots of the same concentrated rhDNase weredialyzed against 1 mM EDTA. One aliquot was then further dialyzed into150 mM NaCl only, and the other into 150 mM NaCl, 1 mM CaCl₂. Afterdialysis, the solutions were adjusted to pH 5 with 1 N HCl. All finalsolutions were filtered and sterile filled into 3 cc glass vials with0.5 mL nominal volume. The stability of these solutions was monitored asabove at 37° C. for up to 6 months for the 0-50 mM CaCl₂ and up to twomonths for >50 mM CaCl₂ samples.

Preparation of 50 mg/mL rhDNase in 1 mM Acetate Buffer. 1, 50 and 100 mMCaCl₂ at pH 5 & 5.2. by Dialysis

rhDNase was concentrated to 50 mg/mL from the bulk rhDNase (4.7 mg/mL)as described above. The concentrated rhDNase was buffer exchanged into 1mM NaOAc, 1, 50 or 100 mM CaCl₂ isotonic with NaCl at either pH 5 or 5.2by dialysis at 2-8° C. for 36 hours. The dialyzed solutions werefiltered and sterile filled into 3 cc glass vials with 0.5 mL nominalvolume. The samples were stored at 37° C. and the stability of rhDNasewas assessed periodically for 1 month with the assays as describedbelow.

Effect of Ca⁺⁺ and Ionic Strength on the Physical Stability of 50 mg/mLrhDNase at pH 5 at 37° C.

50 mg/mL rhDNase solutions at ionic strengths ranging from 0.153 M (150mM NaCl, 1 mM CaCl₂) to 3.15 M (150 mM NaCl, 1 M CaCl₂ and 3.15 M NaCl,1 mM CaCl₂) were prepared by Amicon concentration and dialysis. Thesolutions were adjusted to pH 5 by acid titration. Similarly, anotherset of 50 mg/mL rhDNase solutions buffered at pH 5 with ionic strengthsfrom 0.154 M (1 mM NaOAc, 150 mM NaCl, 1 mM CaCl₂) to 0.226 M (1 mMNaOAc, 75 mM NaCl, 50 mM CaCl₂ and 1 mM NaOAc, 222 mM NaCl, 1 mM CaCl₂)were prepared. The samples were stored at 37° C. and visually inspectedat T=0,1 and 3 days

Determination of Ca⁺⁺ Content in rhDNase Solutions

The Ca⁺⁺ contents were analyzed by Atomic Absorption (AA) according toand by LC (ion chromatography equipped with a conductivity detector.rhDNase samples were diluted with Milli-Q water to 1-5 ppm for AAanalysis and to 30-50 μg/mL for LC analysis. rhDNase in currentformulation at 1 mg/mL (S9847A, 150 mM NaCl, 1 mM CaCl₂), bulk rhDNaseat 4.7 mg/mL, 5.9 mg/mL, and 10 mg/mL, and the concentrated rhDNase at50 mg/mL were analyzed by both methods and compared. rhDNase samplestreated with 1 mM EDTA were also analyzed.

Assay Methods

(1) Color and Clarity: All samples were visually inspected for color andparticulates.

(2) pH: The pH measurement was performed with a Radiometer pHM84 meterequipped with a microelectrode (MI-410, Microelectrodes, Inc.,). Samples(20 uL) were transferred into 0.5 mL eppendorf tubes for measurement atambient temperature.

(3) UV: The concentration of the protein was determined by ultravioletabsorption spectroscopy from 240-400 nm using an HP8451Aspectrophotometer. Appropriate rhDNase excipients were used asreferences. The absorbance at 280 nm was corrected for offset or lightscattering by subtracting the absorbance value at 320 nm. Theconcentration of the protein was determined from the correctedabsorbance at 280 run using an absorptivity of 1.6 cm⁻¹ (mg/mL)⁻¹.

(4) Methyl Green Activity Assay: The activity of the protein wasdetermined by the Methyl Green assay. The samples were diluted to assayrange with assay diluent in duplicate runs. rhDNase reference materialfrozen at −70° C. was used as a reference control and the results werenormalized to this control to account for the inter-assay variation.

(5) Size exclusion chromatography (TSK 2000): To determine the presenceof aggregates and fragments.

(6) Tentacle ion exchange chromatography: To determine the % deamidationin the protein.

(7) SDS-PAGE (Oakley silver stain): To determine the presence offragments and covalent aggregates.

(8) Ca⁺⁺ analysis, (AA) and (LC): To determine the total Ca⁺⁺ content inrhDNase.

Effect of pH on the Stability of High Concentration rhDNase

rhDNase in current formulation (150 mM NaCl, 1 mM CaCl₂) wasconcentrated to ˜50 mg/mL at 2-8° C. with Amicon ultrafiltration using aYM10 membrane. The concentrated solution was clear to slightly yellow.Precipitation occurred upon acid titration to pH 5 but was notnoticeable at pH 6. The solution was clarified by filtration butprecipitated after one day at pH 5 and five days at pH 6 at 37° C. Theprecipitate could be solubilized in strong base. However, in a controlexperiment where there was no precipitation initially, addition ofstrong base to the protein caused denaturation creating high molecularweight aggregates. SDS-PAGE (not shown) of the precipitate confirms thepresence of high molecular weight aggregates and fragments. Both havevery intense bands that spread along the whole lane on the gel.

The pseudo first order kinetics for deamidation of rhDNase at 50 mg/mLin 150 mM NaCl, 1 mM CaCl₂ at pH 5 & 6 (by acid titration) shows thatthe deamidation rate was lower at pH 5 than at pH 6 at higher storagetemperatures (>25° C). See FIG. 7. These pseudo first order rateconstants were in good agreement with the rate constants previouslydetermined for buffered rhDNase solutions at 1 mg/mL (Table 4). Thisresult shows that deamidation of rhDNase is independent of proteinconcentration, but is highly dependent on the pH of the formulation.Since the rate of deamidation is pH dependent, it is important tomaintain the pH of the formulation during storage.

pH variance of 50 mg/mL rhDNase solutions containing various amount ofCa⁺⁺ in 150 mm NaCl, pH 5 at 37° C. for 6 months was determined. SeeFIG. 8. At this temperature, the pH of the formulations with <10 mMCaCl₂ increased gradually with time while the formulations with >1 mMCaCl₂, the pH remained constant. This suggests that increasing Ca⁺⁺ mayprovide a more stable pH at higher storage temperature. The increase inpH of the formulations at 37° C., containing 1 mM or less CaCl₂, may bedue to precipitation of rhDNase. Since the protein is the majorbuffering component in these formulations, a decrease in proteinconcentration may lower the buffering capacity of the formulation. Aformulation where rhDNase was first treated with 1 mM EDTA and thensubsequently dialyzed back into 1 mM CaCl₂ is represented by the symbol0/1 mM CaCl₂ in FIGS. 8 and 9. This formulation should behave the sameas the original formulation of 1 mM CaCl₂.

Effect of Ca⁺⁺ on the Stability of High Concentration rhDNase

Calcium plays a major role in the stabilization of rhDNase. Removal ofCa⁺⁺ by treatment with EDTA resulted in an unstable formulation at 50mg/mL at pH 5 and 37° C. (FIG. 9). The percent of precipitated proteinwas determined indirectly by measuring the decrease in rhDNaseconcentration by UV spectroscopy. The addition of CaCl₂ (up to 200 mM)greatly stabilized the formulation by decreasing the amount ofprecipitated rhDNase (40% vs. ˜2%) even after 150 days at 37° C. Thecloudiness can be distinguished by visual examination. Nevertheless, therates of deamidation for rhDNase at 50 mg/mL are independent of Ca⁺⁺concentrations for up to 6 months at 37° C.

Effect of Direct Acid Titration on rhDNase

After concentration of the bulk rhDNase by ultrafiltration, additional10-200 mM CaCl₂ is dialyzed into the 50 mg/mL rhDNase at 2-8° C. Thedialyzed solutions become turbid during acid titration to pH 5 with HClbut these solutions are less cloudy than the high concentration rhDNaseformulation containing 1 mM CaCl₂ when titrated the same way. Theprecipitates are found to be protein.

It is conceivable that addition of HCl may result in large local pHdecreases before sufficient mixing of the solution is attained. Exposureto local drop in pH decrease may denature some of the rhDNase resultingin the observed precipitation. The denatured rhDNase might also serve asa nucleating species for further association, and ultimately increasedprecipitation over time. The following experiment was set up to studythis matter further. The supernatants of rhDNase solutions at 50 mg/mLcontaining 1 to 200 mM CaCl₂ in 150 mM NaCl, pH 5 (by acid titration)were obtained by centrifugation after the samples were stored at 37° C.for 30 days. The clear supernatants were removed from the originalsolutions, placed into clean glass vials and stored at 37° C. for visualinspection. Small amounts of precipitate were observed only in the 1 mMCaCl₂ formulation after 7 days of storage (Table 5). This suggests thatremoval of denatured rhDNase, which may act as a nucleating site foraggregate formation, inhibits the rate of formation of furtherprecipitate. Increasing Ca⁺⁺ concentration to >1 mM helps to minimizethe denaturation of rhDNase caused by direct acid titration.

The local pH hypothesis was tested by avoiding direct acid titration ofthe formulation. After concentration of the rhDNase with an Amiconstirred cell, the formulation was dialyzed into 150 mM NaCl, 1 mM CaCl₂at pH 4.7. The final rhDNase was a clear solution with a pH of 5.0,whereas, direct titration to pH 5 resulted in a turbid solution. Thisfurther supports the idea that precipitation of protein during acidtitration to pH 5 is due to a local pH effect. No dimers or solubleaggregates were detected by size-exclusion chromatography for the 50mg/mL rhDNase solutions prepared by dialysis at pH 5.

Inclusion of Buffer into the Formulation

A small amount of acetate buffer (1 mM), together with 50 mM or 100 mMCaCl₂ isotonic with NaCl at pH 5 or 5.2, was supplemented into theformulation by dialysis of the concentrated rhDNase in currentformulation. All the buffered solutions with >1 mM CaCl2 remain clearfor up to a month at 37° C. (FIG. 10). The high concentration rhDNase in1 mM NaOAc, 150 mM NaCl, 1 mM CaCl₂, shows a significant decrease in theamount of precipitation upon storage at 37° C. when the pH is increasedfrom 5.0 to 5.2, indicating that the precipitation of protein is very pHdependent. Increasing the level of Ca⁺⁺ to 50 mM or more, results informulations which do not precipitate even after 1 month at 37° C. at pH5 & 5.2. During the preparation of these high concentration rhDNasesolutions, an immediate rise in pH after dialysis is noted especiallyfor the pH 5.2 samples which has a pH of almost 5.4 (FIG. 11). At pH5.2, the 1 mM acetate buffer does not have much buffering capacity, andthe pH rises regardless of calcium concentration.

Table 6 shows that, based on color and clarity, >10 mM CaCl₂ is requiredto maintain the physical stability of the high concentration rhDNase atpH 5 for long term storage at 37° C. A more effective calciumconcentration probably lies in the 25-50 mM range.

Acetate buffers ranging from 0.25 mM to 1 mM was tested to see whichconcentration is adequate for buffering of the 50 mg/mL rhDNasecontaining 1-50 mM CaCl₂ isotonic with NaCl at pH 5 (by dialysis),stored at 37° C. The results are given in Table 7. Based on pH, colorand clarity, the 1 mM NaOAc and 50 mM CaCl₂ formulation gives constantadequate buffering at the desired pH (5.0±0.20) over a period of 28days. There is a slight rise in pH after dialysis. Therefore, the bufferused to prepare high concentration rhDNase should begin with the samebuffer at 0.1-0.2 pH unit lower than the expected pH in the finalformulation.

Effect of Ionic Strength on the Physical Stability of High ConcentrationrhDNase

The protective effect of addition of calcium (to prevent precipitationof protein at 37° C.) beyond 1 mM could be the result of high ionicstrength rather than a specific calcium effect. Table 8 shows that thisprotective effect can be achieved at very high ionic strength (I=3.1 5M) in the formulation such as 1 M CaCl₂ and 150 mM NaCl or 3.15 M NaCland 1 mM CaCl₂ alone. For an isotonic solution at a reasonable ionicstrength, increasing Ca⁺⁺ to 50 mM is definitely beneficial to theformulation at high concentration.

Ca⁺⁺ Binding Study of rhDNase

rhDNase in final vial 1 mg/mL, in bulks 5.9 mg/mL; 10 mg/mL; 4.7 mg/mL,and concentrated rhDNase 50 mg/mL in current formulation of 150 mM NaCl,1 mM CaCl₂ were submitted for calcium analysis by AA and LC (ionchromatography equipped with a conductivity detector). The concentratedrhDNase supplemented with 10-200 mM CaCl₂ and the EDTA treated rhDNasewere also analyzed. The expected calcium concentration would range from0-200 mM. The results (Table 9) show both AA and LC methods arecomparable. One sample (10 mg/mL shown with an asterisk) was alsoanalyzed by Inductive Coupled Plasma—Atomic Absorption Spectroscopy(ICP-AAS) for total calcium. The results are in good agreement with theAA method.

One notable observation is the excess of Ca⁺⁺/mole of rhDNase found inall cases. In all the formulations, regardless of rhDNase and Ca⁺⁺concentration, there are always 4 to 5 more moles of Ca⁺⁺/mole ofrhDNase than expected (Table 9). The EDTA treated rhDNase still has1-1.5 moles of Ca⁺⁺/mole of rhDNase. This suggests there is one tightlybound Ca⁺⁺ to the protein, and the remaining represents weakly boundCa⁺⁺. The 0/1 mM CaCl₂ formulation (EDTA treatment followed by dialysisversus 1 mM CaCl₂) is found to contain 1 mole of Ca⁺⁺/mole of rhDNaseless than the 1 mM CaCl₂formulation. Therefore, the physical stabilityof the 0/1 mM CaCl₂ formulation lies somewhat in between the 1 mMCaCl₂formulation and the one treated with EDTA. A possible formulationfor the portable ultrasonic nebulizer is 40-50 mg/mL rhDNase in 1 mMNaOAc, 30 mM CaCl₂, 105 mM NaCl, pH 5.3.

TABLE 4 First Order Rate Constants for Deamidation in rhDNase asAssessed by Tentacle Ion Exchange Chromatography 1 mg/mL rhDNase 50mg/ml rhDNase 5 mM buffer^(*), 150 mM NaCl 150 mM NaCl 1 mM CaCl₂ 1 mMCaCl₂ pH 5 pH 6 pH 5 pH 6 25° C. 7 ± 1.6 ± 8.5 ± 2.1 ± 1 × 10⁻⁴ 0.1 ×10⁻³ 0.6 × 10⁻⁴ 0.1 × 10⁻³ 37° C. 3.7 ± 9.96 ± 4.9 ± 1.02 ± 0.2 × 10⁻³0.2 × 10⁻³ 0.2 × 10⁻³ 0.05 × 10⁻² *Acetate, pH 5 Succinate, pH 6

TABLE 5 Visual Inspection of the Supernatants of 50 mg/mL rhDNase in 150mM NaCl, 1-200 mM CaCl₂, pH 5 after Centrifugation and Storage at 37° C.Sample * 50 mg/mL rhDNase 150 mM NaCl Color/Clarity at 37° C. pH 5 T =0, 2, 5 days T = 7 days  1 mM CaCl₂ co/cl    co/sl opa 10 mM CaCl₂ co/clco/cl 20 mM CaCl₂ co/cl co/cl 50 mM CaCl₂ co/cl co/cl 100 mM CaCl₂ co/cl co/cl 200 mM CaCl₂  co/cl co/cl * 50 mg/mL rhDNase in 150 mM NaCl,1 mM CaCl₂, was prepared by Amicon ultrafiltration. The 10-200 mM CaCl₂was incorporated into the formulation by dialysis and the finalsolutions were acid titrated to pH 5 with HCl. The samples were storedat 37° C. for more than 30 days. The supernatants of these samples wereobtained after centrifugation at 13,000 rpm for 10 min at ambienttemperature. Keys: co = colorless, cl = clear with no particulate, slopa = slightly opalescent

TABLE 6 pH and Color & Clarity of 50 mg/mL rhDNase in unbuffered CaCl₂isotonic with NaCl. pH 4.7 at 37° C. Sample Day Color/Clarity pH 1 mMCaCl₂, 150 mM NaCl 0 0/0 5.06 28 0/3 5.28 10 mM CaCl₂, 135 mM NaCl 0 0/04.94 28 0/3 5.02 25 mM CaCl₂, 112 mM NaCl 0 0/0 5.31 28 0/0 5.36 50 mMCaCl₂, 75 mM NaCl 0 0/0 5.01 28 0/0 5.05 Keys: Color: 0 = colorlessClarity: 0 = clear 1 = opalescent 2 = slightly cloudy 3 = cloudy

TABLE 7 pH, Color & Clarity of 50 mg/ml rhDNase in 0.25-1 mM Acetatebuffer, 1-50 mM CaCl₂, isotonic with NaCl, pH 5 at 37° C. Sample DayColor/Clarity pH 0.25 mM NaOAc, 0 0/0 5.55 1 mM CaCl₂, 150 mM NaCl 280/3 5.63 0.25 mM NaOAc, 0 0/0 5.54 10 mM CaCl₂, 135 mM NaCl 28 0/0 5.590.25 mM NaOAc, 0 0/0 5.37 25 mM CaCl₂, 112 mM NaCl 28 0/0 5.43 0.25 mMNaOAc, 0 0/0 5.23 50 mM CaCl₂, 75 mM NaCl 28 0/0 5.28 0.5 mM NaOAc,1 mM0 0/0 5.25 CaCl₂, 150 mM NaCl 28 0/3 5.41 0.5 mM NaOAc, 0 0/0 5.24 10 mMCaCl₂, 135 mM NaCl 28 0/1 5.28 0.5 mM NaOAc, 0 0/0 5.37 25 mM CaCl₂, 112mM NaCl 28 0/0 5.43 0.5 mM NaOAc, 0 0/0 5.31 50 mM CaCl₂, 75 mM NaCl 280/0 5.36 1 mM NaOAc, 1 mM 0 0/0 5.14 CaCl₂, 150 mM NaCl 28 0/3 5.34 1 mMNaOAc, 10 mM 0 0/0 5.18 CaCl₂, 135 mM NaCl 28 0/1 5.23 1 mM NaOAc, 25 mM0 0/0 5.25 CaCl₂, 112 mM NaCl 28 0/0 5.30 1 mM NaOAc, 50 mM 0 0/0 5.10CaCl₂, 75 mM NaCl 28 0/0 5.14 Keys: Color: 0 = colorless Clarity: 0 =clear 1 = opalescent 2 = slightly cloudy 3 = cloudy

TABLE 8 Effect of Ca⁺⁺ and Ionic Strength on Precipitation of 50 mg/mLrhDNase at pH 5, 37° C. Ionic Color/Clarity Sample Strength T = 0 T =1,3 days By Acid titration to pH 5: 150 mM NaCl, 1 mM CaCl₂ 0.153 Mco/cl   co/PO 150 mM NaCl, 1 M CaCl₂ 3.15 M co/cl co/cl 3.15 M NaCl, 1mM CaCl₂ 3.15 M co/cl co/cl By dialysis into pH 5 Acetate buffer: 1 mMNaOAc, 150 mM NaCl, 0.154 M co/cl   co/PO 1 mM CaCl₂ 1 mM NaOAc, 75 mMNaCl, 0.226 M co/cl co/cl 50 mM CaCl₂ 1 mM NaOAc, 222 mM NaCl, 0.226 Mco/co   co/PO 1 mM CaCl₂ Keys: 0 = colorless cl = clear PO =precipitates observed

TABLE 9 Comparison of Ca⁺⁺: Determination of rhDNase by LC and AA moleCa/mole rhDNase Ca⁺⁺ (mM) Theoretical Experimental Sample LC AA (AA) 1mg/mL (0.0303 — 1.18 33.00 38.94 mole rhDNase) 150 mM NaCl, 1 1.1 36.30mM CaCl₂ 4.7 mg/mL (0.142 — 1.60 7.04 11.27 mole rhDNase) 150 mM NaCl, 1— 1.63 11.48 mM CaCl₂ 5.9 mg/mL (0.179 — 1.65 5.59 9.22 mole rhDNase)150 mM NaCl, 1 1.80 10.06 mM CaCl₂ 10 mg/mL (0.303 — 2.28 2.33* 3.307.52 mole rhDNase) 7.76* 150 mM NaCl, 1 2.33 2.34* 7.72* 7.39 mM CaCl₂40.83 mg/mL (1.24 1.45 1.73 0 1.40 mole rhDNase) 150 mM NaCl 1.48 1.651.33 41.71 mg/mL (1.26 3.95 3.90 0.79 3.09 mole rhDNase) 150 mM NaCl,0/1 3.83 3.04 mM CaCl₂ 45.96 mg/mL (1.39 5.43 6.03 0.72 4.53 rhDNase)150 mM NaCl, 1 5.60 5.80 4.17 mM CaCl₂ 48.77 mg/mL (1.48 6.23 6.08 0.684.11 mole rhDNase) 150 mM NaCl, 1 6.23 4.21 mM CaCl₂ 43.15 mg/mL (1.3120.00 17.00 7.63 12.78 mole rhDNase) 150 mM NaCl, 10 16.08 16.13 12.31mM CaCl₂ 43.81 mg/mL (1.33 35.00 27.38 15.04 20.59 mole rhDNase) 150 mMNaCl, 20 25.50 26.13 19.62 mM CaCl₂ 45.20 mg/mL (1.37 70.00 57.25 36.5041.79 mole rhDNase) 150 mM NaCl, 50 56.70 55.00 40.15 mM CaCl₂ 51.47mg/mL (1.56 114.15 111.88 64.10 71.72 mole rhDNase) 150 mM NaCl, 100106.25 68.11 mM CaCl₂ 53.50 mg/mL (1.62 211.75 205.00 123.46 126.54 molerhDNase) 150 mM NaCl, 200 mM CaCl₂ ^(a)*EDTA treated ^(b)EDTA treatedand 1 mM CaCl₂ is dialyzed back into the formulation *Analysis by BalcoBay Area Laboratory CoOperative, Burlingame, CA.

Concluding Remarks

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed specific methods usedto prepare and characterize and therapeutically administer theformulation of DNase hereof, and further disclosure as to specific modelsystems pertaining thereto, those skilled in the art will well enoughknow how to devise alternative reliable methods for arriving at the sameinformation in using the fruits of the present invention. Thus, howeverdetailed the foregoing may appear in text, it should not be construed aslimiting the overall scope thereof, rather the ambit of the presentinvention is to be determined solely by the lawful construction of theappended claims.

What is claimed is:
 1. A process for minimizing the aggregation of DNaseat elevated temperatures comprising: introducing a DNaseaggregation-inhibiting amount of calcium cation to a solution comprisingDNase, wherein the temperature of said solution is subsequently elevatedto above 37° C. and aggregation of DNase at said elevated temperature isminimized.
 2. The process according to claim 1 wherein said elevation oftemperature results from spray-drying for collection as a respirableDNase-containing powder that is therapeutically effective whenadministered into the lung of an individual.
 3. A process according toclaim 1, wherein the temperature of said solution is elevated aboveabout 60° C.
 4. A process according to claim 1, wherein said calciumcation is supplied by calcium chloride, calcium oxide or calciumcarbonate.
 5. A process according to claim 1, wherein said calciumcation is present in an amount from 1 mM to 1 M in said solution.
 6. Aprocess according to claim 5, wherein said calcium cation is supplied bycalcium chloride in an amount from 10 mM to 100 mM in said solution.